Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources

ABSTRACT

Methods and systems for real-time monitoring of optical signals from arrays of signal sources, and particularly optical signal sources that have spectrally different signal components. Systems include signal source arrays in optical communication with optical trains that direct excitation radiation to and emitted signals from such arrays and image the signals onto detector arrays, from which such signals may be subjected to additional processing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Provisional U.S. Patent ApplicationNo. 60/772,908, filed Feb. 13, 2006, and to U.S. patent application Ser.No. 11/483,413, filed Jul. 5, 2006, the full disclosures of each ofwhich are hereby incorporated herein by reference in their entirety forall purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Portions of this invention were made under NHGRI Grant No. ROIHG003710-01, and the government may have rights to such inventions.

BACKGROUND OF THE INVENTION

Optical detection systems are generally employed in a wide variety ofdifferent analytical operations. For example, simple multi-well platereaders have been ubiquitously employed in analyzing optical signalsfrom fluid based reactions that were being carried out in the variouswells of a multiwell plate. These readers generally monitor thefluorescence, luminescence or chromogenic response of the reactionsolution that results from a given reaction in each of 96, 384 or 1.536different wells of the multiwell plate.

Other optical detection systems have been developed and widely used inthe analysis of analytes in other configurations, such as in flowingsystems, i.e., in the capillary electrophoretic separation of molecularspecies. Typically, these systems have included a fluorescence detectionsystem that directs an excitation light source, e.g., a laser or laserdiode, at the capillary, and is capable of detecting when a fluorescentor fluorescently labeled analyte flows past the detection region (see,e.g., ABI 3700 Sequencing systems, Agilent 2100 BioAnalyzer and ALPsystems, etc.)

Still other detection systems direct a scanning laser at surface boundanalytes to determine where, on the surface, the analytes have bound.Such systems are widely used in molecular array based systems, where thepositional binding of a given fluorescently labeled molecule on an arrayindicates a characteristic of that molecule, e.g., complementarity orbinding affinity to a given molecule (See, e.g., U.S. Pat. No.5,578,832).

Notwithstanding the availability of a variety of different types ofoptical detection systems, the development of real-time, highlymultiplexed, single molecule analyses has given rise to a need fordetection systems that are capable of detecting large numbers ofdifferent events, at relatively high speed, and that are capable ofdeconvolving potentially complex, multi-wavelength signals. The presentinvention meets these and a variety of other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention is generally directed to systems and methods formonitoring a number of different optical signals from a number ofdifferent and discrete sources of such signals. The methods and systemsare particularly useful in monitoring chemical and biochemical reactionsof interest from an array of reaction regions on a substrate where suchreactions are taking place. Of particular interest are the use of thesemethods and systems in such analytical operations involving relativelyhigh speed, low level signal generation as is found in single moleculeanalyses, e.g., in nucleic acid sequencing reactions.

In one aspect, the invention provides methods and systems for monitoringone or more optical signals from a substrate having at least a firstsignal source disposed thereon. The methods typically comprise imagingthe optical signal onto an imaging detector that comprises a pluralityof pixels. The signal data from a first set of pixels is then subjectedto a first data process, wherein the first set of pixels correspond toat least a portion of the imaged signal. The signal data from a secondset of pixels different from the first set of pixels is subjected to asecond data process different from the first data process. The output ofat least the first data process is then recorded to monitor the opticalsignal.

Relatedly, the systems of this aspect of the invention comprise asubstrate having at least a first source of optical signals disposedthereon, an optical train positioned to receive optical signals from theat least first source of optical signals and image the optical signalsonto a imaging detector, an imaging detector comprising a plurality ofpixels, the detector positioned to receive the image of the opticalsignals on a first set of pixels in the plurality of pixels, and aprocessor programmed to process signal data from the detector to monitorthe optical signals. In accordance with this aspect of the invention, atleast one of the detector or processor are configured to process signaldata from the first set of pixels in a first data process and data froma second set of pixels in the plurality of pixels different from thefirst set of pixels in a second data process different from the firstdata process.

In another aspect, the invention again provides methods and systems formonitoring an optical signal from a source of optical signals. Themethods of this aspect of the invention comprise imaging the opticalsignal onto a plurality of pixels on an imaging detector, followed bycombining signal data from the plurality of pixels, and processing thecombined signal data to monitor the optical signal.

The systems of this aspect typically comprise a substrate having atleast a first source of optical signals disposed thereon, an opticaltrain positioned to receive optical signals from the at least firstsource of optical signals and image the optical signals onto an imagingdetector, an imaging detector comprising a first plurality of pixels,the detector positioned to receive the image of the optical signals on asecond plurality of a pixels in the first plurality of pixels, and aprocessor programmed to process signal data from the detector to monitorthe optical signals. Again, in this aspect of the invention, at leastone of the imaging detector and processor are configured to combinesignal data from the second plurality of pixels to provide combinedsignal data, and process the combined signal data.

In another aspect of the invention is provided methods and systems formonitoring a plurality of spectrally distinct optical signals from asource of optical signals. The methods of this aspect of the inventiontypically comprise passing the plurality of optical signals through anoptical train that is configured to image each of the plurality ofspectrally distinct optical signals onto an imaging detector, wherein animage of each spectrally distinct optical signal has an image shapecharacteristic of its spectral characteristics. The plurality of opticalsignals is then imaged onto the imaging detector. Each optical signal isthen identified by its characteristic image shape to monitor theplurality of spectrally distinct optical signals.

The systems of this aspect of the invention typically comprise asubstrate having at least a first source of optical signals disposedthereon, the source of optical signals including a plurality ofspectrally distinct optical signals. Also included is an optical trainpositioned to receive the plurality of different optical signals fromthe at least first source of optical signals and differentially imageeach of the plurality of different optical signals onto an imagingdetector such that an image of each of the different optical signals ischaracteristic of the spectrally distinct optical signal, an imagingdetector, and a processor for processing signal data from the imagingdetector, wherein the processor is configured to identify the spectrallydistinct optical signal by its characteristic image shape on the imagingdetector.

In still other aspects, the invention provides methods and systems forprocessing an optical image on an imaging detector from a source ofoptical signals. The methods of this aspect of the invention compriseimaging the optical signal onto an array of pixels on an imagingdetector. Signal data is then acquired from the plurality of pixels uponwhich the optical signal is imaged. The acquired signal data is thentransferred to a storage region of the detector, and subjected to a gainprocess during the transferring step to amplify the signal data.

The systems of this aspect of the invention typically comprise asubstrate having at least a first source of optical signals disposedthereon. Also included are an optical train positioned to receiveoptical signals from the at least first source of optical signals andimage the optical signals onto an imaging detector and an imagingdetector. Typically, such imaging detector includes a plurality ofoptically active pixels in an image acquisition portion of the detector,and a data storage portion of the detector operably coupled to the imageacquisition portion to receive signal data from the image acquisitionportion in a frame transfer process. The detector is configured to applya gain voltage to the signal data during the frame transfer process toamplify the signal data transferred to the data storage portion of thedetector.

In another aspect, the invention provides methods, detectors and systemsuseful in monitoring optical signals. The methods of this aspect of theinvention comprise imaging the optical signals onto an imaging detectorthat comprises a plurality of pixels. Signal data from the plurality ofpixels that falls within a selected signal amplitude range is thenselected, and is subjected to a gain protocol to amplify the selectedsignal data, while not amplifying signal data that was not selected.

Relatedly, the invention provides an imaging detector for carrying outthe foregoing method. The detector typically includes a plurality ofoptically active pixels in an image acquisition portion of the detector,a data storage portion of the detector operably coupled to the imageacquisition portion to receive signal data from the image acquisitionportion in a frame transfer process, and a gain register operablycoupled to the data storage portion to amplify signal data from the datastorage portion. The detector is configured to pass signal data throughthe gain register that falls within a selected signal amplitude range.

The invention also provides methods and systems for monitoring opticalsignals where the system comprises a source of optical signals, anoptical train positioned to receive the optical signals from the sourceof optical signals and image the optical signals onto an imagingdetector, and an imaging detector positioned to receive imaged opticalsignals onto a plurality of optically sensitive pixels that are operablycoupled to a gain register to amplify signal data from the plurality ofpixels. The method typically comprises measuring a gain from the gainregister in the absence of an imaged optical signal on the plurality ofoptical signals.

In yet another aspect, the invention provides methods of monitoring aplurality of spectrally different optical signals from a single signalsource. These methods typically comprise collecting the spectrallydifferent optical signals in an optical train. The spectrally differentoptical signals are then transmitted through the optical train that isconfigured to differentially image each of the spectrally differentoptical signals onto an imaging detector. Each spectrally differentsignal imaged upon the detector is then identified by its image on theimaging detector.

In other aspects, the invention provides methods of monitoring opticalsignals from a plurality of signal sources on a substrate, that compriseimaging the plurality of signal sources onto an imaging detector thatcomprises a plurality of pixels, wherein images of the plurality ofsignal sources are directed substantially onto a first subset of theplurality of pixels, but not substantially on a second subset of theplurality of pixels. The signal data from the first subset of pixels butnot from the second subset of pixels are then subjected to further dataprocessing to monitor signals from the plurality of signal sources.

Also provided are methods of monitoring one or more signals from asignal source, comprising: imaging a first optical signal onto a firstplurality of pixels on an imaging detector; selecting from the firstplurality of pixels a first subset pixels that meet or exceed a signalquality threshold; and processing data from the first subset of pixelsto monitor the one or more signals from the signal source.

Other methods of the invention for monitoring one or more opticalsignals from one or more discrete signal sources, comprise: imaging theone or more signals onto a plurality of pixels on a detector array;selecting a subset of the plurality of pixels; recording data from thesubset of the plurality of pixels as indicative of the one or moresignals; correlating the data to a signal from the discrete signalsource.

In still other methods of the invention, a plurality of optical signalsfrom one or more discrete signal sources are monitored. These methodscomprise imaging the plurality of signals onto a detector array, whereineach signal is imaged onto a plurality of pixels on the detector array;and processing data from the plurality of pixels while discarding datafrom pixels not in the plurality of pixels.

The invention also includes systems that comprise: an array of opticalsignal sources, each signal source being capable of emitting a pluralityof signals having different optical wavelengths; an optical train forcollecting the signals from the array of signal sources anddifferentially imaging each of the plurality of signals having differentoptical wavelengths onto a detector; and a detector for detecting thesignals imaged thereon.

The invention is also directed to systems, that comprise an array of aplurality of optical signal sources, the plurality of optical signalsources having a plurality of spectrally resolvable fluorescentcompounds associated therewith. The system also includes a source ofexcitation radiation, a detector array, and an optical train that isconfigured to direct excitation radiation from the source of excitationradiation to the array of signal sources, receive emitted fluorescentsignals from the array of signal sources, and image the fluorescentsignals onto the detector array, wherein the optical train ischaracterized by a dichroic filter in optical communication with anobjective lens, wherein the dichroic filter is reflective of thefluorescent signals and transmissive to the excitation radiation.

In still additional aspects, the invention provides methods forresolvably detecting a plurality of spectrally different optical signalsfrom at least a first signal source, comprising: collecting theplurality of spectrally different optical signals from the signalsource; and differentially imaging each of the spectrally differentoptical signals on a detector array, such that each different signal isresolvably detected.

Relatedly, the invention also provides methods of monitoring an opticalsignal from a signal source, comprising: imaging the optical signal ontoa plurality of pixels of a detector array in a signal image; selecting asubset of the plurality of pixels in the signal image having a highersignal intensity within the signal image than other pixels within thesignal image; and measuring the signal in the subset of pixels.

Also provided herein are methods of monitoring signals from a pluralityof signal sources, comprising: imaging each of the plurality of signalsonto a detector array comprising a plurality of rows and columns ofpixels; processing data derived from pixels in rows or columns uponwhich the plurality of signals is imaged, but not from rows of pixelsupon which no signal is imaged.

Other methods of the invention for processing signals imaged onto anEMCCD, comprise: determining whether the signals imaged onto the EMCCDare within a preselected signal amplitude range; and processing onlysignals that are within the preselected amplitude range through a gainregister on the EMCCD.

In still other methods of processing signals imaged onto a CCD that isconfigured to transfer image data acquired by the detector to a storageregion on the CCD in a frame transfer process, an elevated voltage isapplied to the frame transfer process to amplify signal data beingtransferred.

In a further aspect, the invention provides systems for monitoringoptical signals from a plurality of sources of optical signals,comprising: an array of discrete sources of optical signals, saiddiscrete sources emitting optical signals having different spectralcharacteristics; an excitation radiation source; a detector array; anoptical train configured to: direct excitation radiation from the sourceof excitation radiation to the array of discrete sources; receiveemitted optical signals from the array of signal sources; anddifferentially image the optical signals having different spectralcharacteristics onto a detector array; and a processor configured torecord the optical signals imaged onto the detector array and correlatethe optical signals with a property of or an event occurring within thesources of optical signals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of an overall system the presentinvention.

FIG. 2 provides a schematic illustration of an array of signal sourceson a substrate, such as zero mode waveguides.

FIG. 3 provides a schematic illustration of an alternative spacingand/or orientation scheme for an array of signal sources, in accordancewith certain aspects of the invention.

FIG. 4 is a schematic illustration of one example of a mounting stageconfigured to receive and support substrates bearing signal sources foranalysis in the systems of the invention.

FIG. 5 is a schematic representation of an x-y-z translation roboticsystem for translating one or more of the substrate and/or the opticaltrain relative to the other, within the systems of the invention.

FIG. 6 schematically illustrates the substrate and optical train of thesystems of the invention that includes optical componentry for theseparation and detection of spectrally resolvable signal components.

FIG. 7 provides a schematic illustration of a system of the presentinvention that includes optical componentry for simultaneousillumination of larger numbers of signal sources on the substrates.

FIG. 8 provides a schematic illustration of a multiple excitationsource/multiple emission wavelength system that utilizes transmissivefluorescence optics.

FIG. 9 provides a schematic illustration of a multiple excitationsource/multiple emission wavelength system that utilizes reflectivefluorescence optics.

FIG. 10 provides a block diagram illustrating the operation of an EMCCDdetector and data processing steps of certain aspects of the invention.

FIG. 11 provides a comparative illustration of signal image correlationmethods from a detector array that take into account optical aberrationsin the upstream optical train.

FIG. 12 schematically illustrates a comparison of data extractionprocesses in conventional image processing versus processes employed incertain aspects of the invention.

FIGS. 13A and B schematically illustrate pixel correlation to imagedsignals or signal components to improve the fidelity of data from agiven image or set of images.

FIG. 14 schematically illustrates data management on an EMCCD detectorto enhance efficiencies of the system.

FIG. 15 provides a flowchart of data processing from CCD detector arraysto minimize effects of large signal variations.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is generally directed to optical detection ormonitoring systems, methods enabled by such systems, and components ofsuch systems for monitoring, in real-time, optical signals that emanatefrom multiple discrete sources of those optical signals. In particular,the optical detection and monitoring systems of the invention aregenerally capable of monitoring discrete signals from potentially verylarge numbers of different signal sources, optionally separating and/ordeconvolving such signals into constituent signal events, and doing soin real-time, despite that such signals may be changing rapidly, overtime.

The systems of the invention thus include all or a portion of acollection of different functional elements. These elements include themultiple discrete sources that include the capability of generatingoptical signals. In preferred aspects, such sources include chemical,biochemical and/or biological reactants, or mimics of such reactantsthat are capable of generating optical signals that are indicative oftheir presence, reaction or conversion. While the sources may be capableof generating optical signals on their own, in preferred cases, a sourceof excitation radiation is also provided to excite optical signals,e.g., fluorescence, within the sources.

The systems of the invention also typically include optical elementsthat direct, separate, and/or otherwise alter optical signals from thesesources (as well as excitation radiation directed at such sources), inorder to ultimately derive optimal amounts of information from suchsignals when they are ultimately detected. Consequently, the systems ofthe invention typically include an optical detection system fordetecting the potentially large numbers of signals that were directedfrom the sources, and optionally separated and/or otherwise altered bythe optical elements.

Signals detected by the optical detection system are then recorded andprocessed by appropriate processing systems and data managementprocesses to provide output of the system in user ready formats.

As alluded to previously, the systems of the invention are preferablyapplied in the monitoring of arrays or collections of spatially discretechemical, biochemical and/or biological reactions that generateoptically detectable signals, such as chromogenic reactions, luminescentor luminogenic reactions, or fluorescent or fluorogenic reactions. A fewexamples of preferred reactions include those that are regularlyperformed in the pharmaceutical, biotechnology and healthcare diagnosticfields, i.e., immunoassays, enzymatic assays, receptor assays, nucleicacid hybridization assays, nucleic acid synthesis reactions, cellularassays, and many others.

Typically, the progress of the reactions used in application of thesystems described herein result in one or more of the consumption,production and/or conversion of a material that is capable of generatingan optically detectable signal, either alone, or in response to anexternal stimulus, e.g., excitation radiation. By way of example,certain reactants may become fluorescent upon reaction with anotherreactant, or may have their fluorescence altered or reduced upon suchreaction. As such, the fluorescence emitted from the reaction inresponse to an excitation radiation will change as the reactionprogresses. The systems of the invention provide for the source of suchsignals, e.g., the area in which the reaction occurs, includingoptionally, the reactants and/or products, the optical elements forcollecting, directing and optionally separating and/or altering suchsignals from such sources, and the ultimate detection of such signals,as well as the manipulation of the resulting data to yield optimal valueand information for the user.

The systems of the invention typically include all or a subset of asubstrate that includes all or a subset of the sources of opticalsignals, an optional excitation light source, an optical train thatincludes the various optical elements for collection, direction and/ormanipulation of the optical signals and optional excitation light,optical detectors for receiving, detecting and recording (or puttinginto a form for recordation) the optical signals, as well as processorsfor processing data derived from the optical detectors.

A general schematic representation of the system as set forth above, isillustrated in FIG. 1. As shown, the system 100 includes a substrate 102that includes a plurality of discrete sources of optical signals, e.g.,reaction wells or optical confinements 104. An excitation light source,e.g., laser 106, is optionally provided in the system and is positionedto direct excitation radiation at the various signal sources. This istypically done by directing excitation radiation at or throughappropriate optical components, e.g., dichroic 108 and objective lens110, that direct the excitation radiation at the substrate 102, andparticularly the signal sources 104. Emitted signals from source 104 arethen collected by the optical components, e.g., objective 110, andpassed through additional optical elements, e.g., dichroic 108, prism112 and lens 114, until they are directed to and impinge upon an opticaldetection system, e.g., detector array 116. The signals are thendetected by detector array 116, and the data from that detection istransmitted to an appropriate data processing unit, e.g., computer 118,where the data is subjected to interpretation, analysis, and ultimatelypresented in a user ready format, e.g., on display 120, or printout 122,from printer 124.

The various functions, applications and components of the systems of theinvention are set forth in greater detail below.

II. Substrate

A. Substrate

As alluded to previously, the substrates of the invention, as a generalmatter, provide the multiple discrete sources of optical signals. In thecase of systems for monitoring reactions, such signal sources typicallycomprise discrete regions in which reactions are taking place and fromwhich discrete optical signals may emanate. In a broad sense, suchdifferent regions may comprise reaction wells, or zones that aremaintained discrete from other regions by any of a number of differentmechanisms, including chemical or physical confinements. Merely by wayof example, such regions may comprise discrete patches or zones ofimmobilized molecules on a surface of the substrate, such as in nucleicacid, protein, antibody or other immuno-arrays, where the reaction beingmonitored is the association of analytes with such immobilizedmolecules, they may include channels within a substrate, e.g.,microfluidic channel regions, aggregations of capillaries or multipleregions within individual capillaries, or the like.

Alternatively or additionally, such regions may include structuralconfinements that maintain the reaction components within the discreteregions. Such structural confinements may include wells, depressions,channels, or other structures that retain reaction constituents. Suchconfinements may also include other bathers that effectively providestructural confinement through, e.g., the use of chemical barriers,e.g., hydrophobic regions surrounding hydrophilic regions on thesubstrate surface to retain aqueous reaction constituents within thehydrophilic regions.

In still other aspects, such regions may include combinations of theabove, e.g., including immobilized reactants within structuralconfinements. In addition to structural confinements, the reactionregions may comprise optical confinements that may function as or inaddition to structural confinements on the substrates, that serve tominimize observation volumes on the substrate through the confinement ofexcitation illumination and/or the collection of emitted optical signalsfrom relatively small areas or volumes at the reaction region. Suchoptical confinements may include, e.g., waveguides, such as zero modewaveguides, optical gratings, optical coatings or the like, that canyield the excitation or observation volumes desired on the reactionregions on the substrates.

Typically, the substrates will comprise an optically transparent layerupon which are disposed the reaction regions that provide the discretesources of optical signals. The optically transparent layer maygenerally comprise any of a number of transparent solid materials,depending upon other components of the substrate. Such materials includeinorganic materials, such as glass, quartz, fused silica, and the like.Alternatively, such materials may include organic materials, such aspolymeric substrates such as polystyrene, polypropylene, polyethylene,polymethylmethacrylate (PMMA), and the like, where PMMA is particularlyuseful in fluorescent or fluorogenic reactions, as it has relatively lowautofluorescence.

In preferred aspects, the substrates include zero mode waveguides as theoptical confinements to define the discrete reaction regions on thesubstrate. Zero mode waveguides have been described in, e.g., U.S. Pat.No. 6,917,726, the full disclosure of which is incorporated herein byreference in its entirety for all purposes. Briefly, such waveguidescomprise a core disposed through a cladding layer, which in the case ofapplications to reactions, comprises an aperture disposed through thecladding layer that can receive the reactants to be monitored.Typically, the aperture has at least one cross-sectional dimension,e.g., diameter, which is sufficiently small that light entering thewaveguide is prevented in some measure from propagating through thecore, effectively resulting in a very small portion of the core and itscontents being illuminated, and/or emitting optical signals that exitthe core. In the case of optical signals (and excitation radiation), thewaveguide cores will typically be between 1 nm and 200 nm, and arepreferably between about 10 and 100 nm, and more preferably betweenabout 30 and about 100 nm in diameter.

Optical confinements are typically provided upon the substrate in anarray format where a plurality of confinements are provided upon thesubstrate. In accordance with the invention, arrays of confinements,e.g., zero mode waveguides, are provided in arrays of more than 100,more than 1000, more than 10,000, or even more than 100,000 separatewaveguides on a single substrate. In addition, the waveguide arraystypically comprise a relatively high density of waveguides on thesurface of the substrate. Such high density typically includeswaveguides present at a density of greater than 10 zero mode waveguidesper mm², preferably, greater than 100 waveguides per mm² of substratesurface area, and more preferably, greater than 500 or even 1000waveguides per mm² and in many cases up to or greater than 100,000waveguides per mm². Although in many cases, the waveguides in the arrayare spaced in a regular pattern, e.g., in 2, 5, 10, 25, 50 or 100 ormore rows and/or columns of regularly spaced waveguides in a givenarray, in certain preferred cases, there are advantages to providing theorganization of waveguides in an array deviating from a standard rowand/or column format.

Optical systems often include a number of optical aberrations,including, e.g., astigmatism, chromatic aberrations, coma, distortion,field curvature, and spherical aberration. In many instances, theseoptical aberrations become more pronounced as a function off distancefrom the axial center of the optical imaging system, such that themagnitude of the aberration varies as a function of field position.Accordingly, the optical image is typically most free of aberrations ator near the center of the object field, and is more distorted at theperiphery of the object field and system pupil. Because of suchaberrations, resolution and accurate monitoring of arrays of discretenanometer or micron scale sources of optical signals that are providedin a relatively high density becomes increasingly problematic away fromthe center of the object field. Consequently, performing analyses in ahighly multiplexed array of waveguides or other signal sources becomesmore difficult.

In accordance with one aspect of the invention, therefore, the sourcesof discrete optical signals, e.g., the optical confinements, i.e., zeromode waveguides, in array formats, are arranged within the array in anon-regular format, to account to minimize the impact of these expectedoptical aberrations, and as a result permit more effective multiplexedanalyses. In particular, individual sources of signal in the array maybe positioned to account for reduced resolution, e.g., betweenneighboring sources, as a function of distance from the center of theobject image. Additionally, or alternatively, the discrete sources maybe dimensioned to account for reduced resolution and accuracy at theperiphery of the object field. The variance in optical resolution, orconversely, aberration, as a function of distance from the center of theobject field are particularly noteworthy in systems that rely uponimaging based detection systems, e.g., that effectively image an entirearray or region of an array, that includes multiple different signalsources. Examples of such systems include detector arrays, such as diodearrays, CCDs, i.e., ICCDs and EMCCDs, and/or CMOS based image sensors,where signals are detected at individual or small groups of pixels onthe detector. For example, in CCD based detectors, as signals becomemore distorted away from the axial center of the imaging system, itbecomes increasingly difficult to assign pixel areas on the CCD thatcorrespond to a given signal source in the array of signal sources.

FIGS. 2 and 3 show a comparative illustration of arrays of sources ofoptical signals. FIG. 2 shows an array 200 of sources of optical signals(shown as an array of zero mode waveguides 204 in a substrate 202) thatincludes regularly spaced and consistently sized sources of opticalsignals. As noted previously, in some cases the sources at the peripheryof the array, e.g., sources 206 and 208 would be less resolved,optically, than, e.g., sources 210 and 212. In some cases, it may be thecase that aberrations could be sufficient to prevent resolution of theperipheral sources, e.g., 206 and 208. Accordingly, as shown in FIG. 3,an array 300 of sources 302 is provided where the spacing betweenadjacent sources is increased as a function of the distance from thecenter of the object image. For example, as shown, signal sources thatare nearer the center of the object field represented by the array 300,e.g., sources 304 and 306 are closer together in at least one dimension,than are sources that further away from the center of the object field,e.g., sources 308 and 310, which are more widely spaced in at least onedimension than the more central signal sources. Thus, the space, in atleast one dimension between two sources at a first distance from thecenter of the object field of the optical system will be less than thespace, again in at least one dimension, between two sources that are ata second, greater distance from the center of the object field. Thespacing between adjacent signal sources may be varied in only onedimension, e.g., varied from left to right, but not from top to bottom,or it may be varied in both dimensions. In the case where the spacing isvaried in both dimensions, it will be appreciated that the distancebetween any two signal sources at the center of the object field, e.g.,is less than the space between any two signal sources further away fromthe center, i.e., on the periphery, of the object field. The foregoingpermits greater effective multiplex analysis of arrays of signalsources, such as ZMWs.

Additional arrangements of array elements can be specifically tailoredto fit a particular aberration of particular optical systems. Forexample, if a dominant optical aberration forms a resulting image spotsize or shape that is dependant upon field location, then that size orshape can be accommodated in the design of the array of sources by,e.g., appropriately spacing the sources to avoid overlap in image ofadjacent sources, or the like. Similarly, if the shape of an imagedsource is distorted in one dimension so as to potentially overlap withimages of neighboring sources, that source can be dimensioned to reducethat dimension and avoid the overlap, e.g., providing elliptical orrectangular sources.

In a simpler aspect, the signal sources may also be spaced to accountfor optical manipulations of the signals emanating therefrom. Forexample, as discussed in greater detail below, in some cases, opticalsignals are spatially separated into component elements, e.g., light ofdifferent wavelength ranges, indicative of different signaling elements,i.e., fluorescent reagents having differing emission spectra. In suchcases, it may be desirable to provide sufficient spacing betweenadjacent signal sources on the substrate to prevent overlap of thespatially separated signals derived from those sources, when thoseseparated signals are incident upon the detector, as set forth below. Inthis case, increased spacing may only be required in one dimension,e.g., providing sufficient spacing between rows of signal sources, butnot necessarily between the columns of signal sources in the array.Alternatively, such additional spacing may be provided in twodimensions. In the case of arrays of signal sources where the signalsare subjected to spatial separation before detection, such spacingbetween adjacent signal sources may generally range from about 0.1 μm toabout 10 μm or more, and is preferably from about 0.8 μm to about 3 μmor more.

B. Substrate Interface

The substrates of the invention are typically interfaced with theoverall system through an appropriate mounting stage that secures thesubstrate, provides translational capability to the substrate, e.g.,relative to the optical system, and optionally provides additionalfunctionalities, e.g., fluidic interfaces, thermal regulation, e.g.,heating or cooling, positional registration, and the like. The mountingstage will also typically include a positioning element that ensuresproper positioning and/or orientation of a substrate upon the stage, forsubsequent analysis. Such positioning systems may keyed structures onthe substrate that are complementary to a corresponding structure on themounting stage. These may include simple structures, e.g., tooth/notchstructures, truncated corner structures, or other distinctive andcomplementary structures. Alternatively, the keying elements may includeelectronic keys, such as metal contacts and associated electroniccomponents on the substrate and mounting stage, that indicate when asubstrate is positioned properly and in the correct orientation forsubsequent analysis. Such key elements may be provided encoded for eachsubstrate, e.g., through incorporated memory elements on the substrate,or through the position and orientation of electrical contacts, toindicate a specific substrate, e.g., lot number, etc. Suchidentification systems may provide an ability to ascertain whether agiven substrate has been used previously, and to what effect. Typically,the mounting stage includes a well or recessed component configured toreceive the substrate or the packaged structure containing thesubstrate, e.g., a multiwell plate format, as well as a biasingmechanism, e.g., spring, clip or other mechanism, for forcibly retainingthe substrate in a fixed position on the stage.

One example of a mounting stage is shown in FIG. 4. As shown, themounting stage 400 includes a platform 402 having a mounting region 404that receives the substrate (not shown). The mounting region istypically disposed over an aperture 406 in the platform 402 that allowsobservation of the substrate from underneath. Also as shown, themounting stage includes structures that facilitate the positioning andalignment of the substrate on the platform. These may include, e.g.,ridges 406, recesses or wells, for positioning the substrate, andalignment structures 408, such as pins, bevel structures, tabs, or thelike, that correspond to a complementary structure on the substrate,e.g., holes or notches. As noted above, securing mechanisms may also beprovided for locking the substrate in place, such as biasing mechanism410, shown as a clip or a closable cover element, shown also from a sideview. Additional components may be provided on the mounting stage, suchas a heating or cooling element, additional optical components, andother interfacing elements.

The mounting stage is also typically coupled to a translation system formoving the stage in two or three dimensions relative to the opticalsystem. The translation system allows scanning of the entire array ofsignal sources on a substrate, as well as providing an ability to movethe substrate toward or away from the optical system for, e.g.,focusing, removal of the substrate, addition of components to thesubstrate, or the like. A variety of x-y-z translation systems arereadily available. Additionally, robotic systems are readily availablefor automating the translation functions of the mounting stage inaccordance with preprogrammed instructions. FIG. 5 shows a schematicrepresentation of an entire system 500 including a schematicallyrepresented translation system 502 coupled to a mounting stage 504,supporting substrate 506 over optical train 508. As shown, the roboticsystem includes the capability to move the substrate in any of the x, y,or z dimensions.

Robotic systems may also include components that position substratesupon the mounting stage, apply reagents to the substrates, and the like.A wide variety of such robotic systems that may be applied to thepresent invention are generally commercially available from, e.g.,Tecan, Inc., Caliper Life Sciences, Inc., Beckman, Inc., and the like.

III. Excitation Source

As noted previously, in preferred applications, the systems of theinvention are used to monitor luminescent or fluorescent signalsemanating form the plurality of discrete signal sources. As such, inmany cases, the systems of the invention include a source of excitationradiation. Excitation light sources will generally depend upon thenature of excitation radiation needed for a particular application,e.g., as dictated by the reagents and configuration of a given analysis.For example, the light source may include lamps, e.g., halogen, Mercury,Xenon, or the like, LEDs, lasers, laser diodes, or any other lightsource capable of directing electromagnetic radiation of a desiredexcitation wavelength or wavelength range, to the signal sources on thesubstrate. In preferred aspects, lasers are preferred as the excitationradiation source, due to the coherency and intensity of radiation thatthey generate in desired excitation wavelength ranges. A variety ofdifferent laser types are generally useful for these applications, andinclude, e.g., ion lasers, solid state direct diode lasers, diode-pumpedsolid state lasers (DPSS), solid state frequency converted crystallasers, and the like. In some cases multiple sources may be employed inorder to provide multiple different excitation wavelengths. By way ofexample, in cases where the signal sources include fluorescentcompounds, e.g., compounds labeled with fluorescent dyes, multipledifferent excitation sources may be provided for the various differentexcitation spectra for such compounds. For example, in the case ofcompounds labeled with Alexa648 dyes, it will typically be desirable toprovide at least an excitation source that provides excitation radiationrange that includes light at the 648 nm, the respective excitationwavelengths for these dyes, or if not provided at the nominal peak ofthe dye absorbtion curve, the lasers will include sufficient absorbtionefficiency for the dyes used, such as for Alexa546, where the peakabsorption efficiency is closer to 561 nm. In the cases of multipledifferent dyes, different lasers, e.g., having different wavelengthranges may be used.

IV. Optical Train

As noted previously, the overall systems of the invention typicallyinclude an optical train for the direction of excitation radiation tothe substrate and the plurality of signal sources thereon, and/or fordirecting emitted signals from these sources to a detection system thatquantifies and records the signal from each signal source. The opticaltrains used in the overall systems described herein typically include anumber of different optical components for use in focusing, directing,splitting, separating, polarizing, and/or collimating the excitationradiation and/or the signals emanating from the discrete sources ofsignals.

A schematic illustration of one optical train is shown in FIG. 6. Asshown, the optical train includes an objective lens 602 that is proximalto the substrate 604, and which focuses excitation radiation, e.g., fromlaser 606, upon a desired location of the substrate, and collectsemitted optical signals from the substrate. The optical train will alsotypically include one or more dichroic mirrors/filters 608, thatselectively reflect or pass excitation light and emitted opticalsignals, to effectively separate signal radiation from reflected orotherwise errant excitation radiation.

The optical train may also optionally include signal separation optics,e.g., to separate optical signals of different wavelengths or directthem to different locations on a detection system. For example, theoptical train may include prism 610 that receives the optical signs asfrom the signal sources, that may include signals of several differentprimary wavelengths. Alternatively, sets of dichroic filters may be usedin a cascading arrangement, to selectively direct each differentspectral signal component to a different detector or detector region.

In the case of a prism as a separation element, upon passing through theprism 610, the different wavelength signals are diffracted to differentdegrees, and as a result, are directed, optionally through additionaloptical components, i.e., imaging lens 612, at different angles towardthe detection system, e.g., detector array 614 allowing for theirseparate detection and quantitation.

The ability to separate such signals is of particular value inmonitoring signal sources that include multiple different reagents thateach have a different fluorescent emission spectrum, indicative of adifferent specific reagent, reaction and/or interaction. A variety ofother optical components may be employed in spectrally separating theoptical signals, including cutoff filter sets, dichroics, opticalgratings, and the like. Such components will typically be arranged todirect different portions of each optical signal to different detectorsor, preferably, different locations upon the same detector or array ofdetectors. In accordance with the invention, different signals may bespectrally resolved by differentially imaging such signal componentsonto the detector, e.g., detector array 614. Such differential imagingmay be entirely spatially distinct, e.g., by being directed to differentdetectors or locations on the same detector, or they mayconformationally distinct, e.g., providing an imaged signal that is of adifferent shape than an image of a different signal component, such thatit can be resolved. For ease of discussion, both shall be generallyreferred to herein as being spatially resolved or separated or directedto different or regions of the detector, although in some cases, suchdifferent regions will be understood to overlap.

Other components that separate portions of the optical signals are alsooptionally included in the optical train, depending upon the applicationto which the system is to be put, including spatial filters, e.g., toconfine the optical signals that are directed to the detector,polarizing filters, to pass signals that are in one polar optical plane,or the like. For example, in addition to separation of signals ofdiffering wavelengths, the optical train may also include splitters,e.g., beam splitters, optical gratings, lens or microlens arrays, andthe like, that serve to divide up the excitation radiation and/or theemitted signals to direct it to different locations, or other opticalcomponents that change the spatial configuration of excitationradiation, e.g., optional optical grating 616. In some cases, additionalfilters may be added after the laser to filter the main laser line byremoving or reducing any optical noise that may be inherent in thelaser, as well as in front of the detectors to reduce or remove anyunwanted stray light that may be generated or reflected from the systemas a whole, or the ambient light.

In particular, in certain aspects, one or more of the optical trainand/or the excitation radiation source may be configured so as toprovide excitation illumination of a large number of discrete signalsources on the substrate simultaneously. In the case of arrays of zeromode waveguides, for example, the optical train and/or the excitationradiation source provide illumination to a large number of zero modewaveguides, simultaneously. As noted below, the optical trains are alsotypically capable of collecting and detecting signals from the same orsimilar large numbers of the signal sources, or in this example, zeromode waveguides. The systems typically illuminate at least 2 signalsources, simultaneously, preferably, greater than 10 signal sourcessimultaneously, and more preferably, greater than 100 signal sources,simultaneously. In some cases, it may be desirable to use the systemsdescribed herein, for the excitation of 1000, 10,000 or more discretesignal sources. Systems that split excitation beams or apply multipleexcitation sources (both with or without beam splitting) areparticularly useful for directing excitation radiation to larger numbersof signal sources.

Simultaneous illumination with excitation radiation over large numbersof signal sources may generally be accomplished through a variety ofdifferent means, as noted above. For example, one may focus a relativelylarge spot size upon a large array of signal sources. However, as willbe appreciated, because laser power is limited, and indiscriminateillumination may cause certain adverse effects, e.g., heating, it may bedesirable to avoid illuminating non-signal generating portions of thesubstrate. Additionally, in many cases, the non-signal generatingregions of the substrate may provide additional noise through reflectionof the laser light. For example, in the case of arrays of zero modewaveguides using a thin film metal cladding layer, spaces between signalgenerating regions are highly reflective. Such reflected activationradiation gives rise to elevated noise levels for the system.

In some cases, larger excitation regions may be provided by directingmultiple different excitation sources at a given substrate to provideillumination of larger numbers of signal sources, e.g., laser 606 andoptional additional lasers, e.g., as shown in FIGS. 8 and 9.Unfortunately, use of multiple different sources may provide issuesregarding differences between the individual sources, e.g., wavelength,frequency or intensity of illumination that may impact the signalsresulting therefrom, e.g., rendering slightly different signal profiles.Additionally, such multiple excitation source systems may still giverise to the problems of excessive illumination of the substrate, as awhole. Similarly, excitation light beams may be divided into multiplebeams, e.g., using beam splitters, optical gratings or other opticalcomponents, as alluded to above, to direct multiple discrete excitationillumination spots at different locations of the substrate, and as aresult, illuminating larger numbers of signal sources thereon. In arelated aspect, lenses may be provided that stretch the beam spot intoan elliptical or elongated spot shape.

In certain preferred arrangements, individual or multiple excitationradiation source(s) may be manipulated to provide preferentialillumination on the signal sources on a substrate, and reduce oreliminate illumination at regions of the substrate not occupied by thesignal source(s). A number of methods may be used to modulate theillumination profile of the excitation light source to preferentiallyprovide excitation illumination at the signal sources on the substrate,and, in particularly preferred aspects, less illumination at the spacesnot occupied by such signal sources. In general, this is accomplished byusing optical elements that provide a signal profile at the object planeof the optical train, e.g., the substrate, that peaks in intensity atpositions in the object plane that correspond to the position of thesignal sources on the substrate. A variety of different optical elementsmay be used to achieve this illumination profile. For example, whereillumination at a low frequency is not an issue for analysis of thesignal sources, one may simply employ reciprocating beam, e.g., throughthe use of a galvo-equipped laser system. In cases where low frequencyillumination is or can be an issue, one may employ holographic ordiffractive optical elements to achieve the desired illuminationprofile, e.g., in rows of lines, grids, or the like.

In particularly preferred aspects, cylindrical lenses or microlenses, orarrays of cylindrical lenses or microlenses are used to modulate theexcitation light to provide illumination in a linear format so as topreferentially illuminate regions that include signal sources, and donot illuminate regions of the substrate that include no signal sources.Further, such optical elements may yield excitation illuminationprofiles on the substrate in multiple lines, i.e., in parallel and/or inorthogonal orientation, e.g., as a grid, or the like. For purposes ofdiscussion, and with reference to direction at the substrate andincluded arrays of signal sources, the “laser spot” or “excitationradiation spot” refers to any of a variety of different beam shapes,configurations and orientations that are incident upon the substrate,including ellipses, lines, grids, and the like. As will be appreciated,when selectively directing excitation radiation at the signal sources onthe substrate, the system may be equipped with certain alignment toolsto facilitate alignment of the excitation radiation with the arrays ofsignal sources on the substrate. Such tools may include referencepositions on the substrate that may be identified, either manually orautomatically, by the system, to orient and/or focus the systemappropriately on the array of signal sources on the substrate.

A schematic illustration of this aspect of the invention is shown inFIG. 7. As shown, the excitation illumination portion of an overallsystem 700 includes the excitation light source, e.g., laser 702, thatis directed through an appropriate optical element, here shown as anarray of cylindrical lenses 704, to an appropriate dichroic mirror,e.g., dichroic 706, which directs the excitation radiation (shown assolid arrows) up through objective lens 720 and toward substrate 710. Asnoted previously, the spatial profile of the excitation radiation isconfigured to preferentially provide greater excitation radiation at thevarious signal sources 708 on the substrate 710, which is in the focalplane of the objective lens 720. An alternate view of substrate 710shows the illumination profile as a series of parallel illuminationregions on the substrate (as indicated by the dashed outlines 712).

As described elsewhere, herein, the emitted fluorescence or otheroptical signals from the signal sources, are then collected by objective720, passed through dichroic 706, and are optionally subjected tospectral separation of the signal components, e.g., via prism 714, andultimately directed to a detector, e.g., detector array 718. In additionto the various optical components already discussed, the optical trainsof the systems described herein may also include one or more imaginglenses, e.g., lens 716, to provide a resolved image of the separated,and directed optical signals onto an image plane of, e.g., a detectorarray 718.

While linear laser or illumination “spots” are preferably aligned to becollinear with rows and/or columns of spatially arrayed signal sources,it will be appreciated that such illumination lines may be provided atan angle that is offset from the linear arrangement of the signalsources, but still illuminating multiple different signal sourcessimultaneously. In particular, by offsetting the illumination lines by aselected angle, one can still ensure that illumination of multipleregularly arrayed or gridded signal sources are illuminated. In itssimplest form, for example, an illumination line rotated at 45° from thelinear arrangement of signal sources in a grid will still illuminatethose signal sources that lie on the diagonal. Similarly, as withregularly spaced rows of crops passed by on the adjoining roads,numerous specific angles provide linear arrangements of adjacent signalsources. As will be appreciated, the angles that provide effectiveillumination across multiple different signal sources in a gridded arrayformat will generally depend upon the spacing of the sources in eachdimension. For regularly spaced sources, e.g., equally spaced in twodimensions, for example, lines at 0°, 22.5°, 45°, 67.5° and 90° anglesfrom the row or column orientation of the gridded array of signalsources will generally run parallel to lines that include multiplesources. A number of angles between these will likewise provideillumination of multiple sources.

The various components of the optical train, e.g., lenses, gratings,filters, prisms, beam splitters, and the like, are generally obtainablecommercially from optics suppliers, including, for example, SpecialOptics, Inc., Newport Corporation, Thorlabs, Inc., CVI Lasers, LambdaResearch Optics, Lambda Physics, and Precision Optical, Inc.

In some aspects, the optical train for use in the systems of the presentinvention utilizes a configuration based upon reflective fluorescencefilters that more readily permit implementation of multi-light source,e.g., laser, excitation systems, that may be useful formulti-fluorophore systems, e.g., signal sources that include multipledifferent fluorophores in generating the signals.

In conventional fluorescence detection schemes, interference filters aretypically employed that reflect excitation light at an angle ofapproximately 90° such that is incident upon the fluorescent sample, andtransmit fluorescent light emitted from that sample such that itswave-front remains relatively undisturbed. While the degree of rejectedexcitation light attainable in such transmissive fluorescence geometriesis sufficient for most one or two excitation band applications, thesecurrent schemes may not be effectively extended to three or fourexcitation band schemes, as a single transmissive-fluorescence filterthat efficiently passes substantial portions of multiple, e.g., 2, 3, 4or more, different fluorescent spectra while reflecting the multipleexcitation bands, is not readily manufacturable using availabletechnology. Further, while multiple filter components could be combinedto achieve this in a multiple laser, multiple emission wavelengthsystem, increased transmission losses, increased optical aberrations,increased size, and increased costs for making higher performancefluorescence transmissive filter systems, make such solutions lessdesirable.

In contrast, the optical trains of certain preferred configurations ofthe systems of the invention utilize a reflective fluorescence filtersetup in selectively directing emitted light to the detector whileblocking excitation light that is reflected from the substrate or othercomponents in the system. In particular, the optical trains of thisaspect of the invention typically include at least one optical filtercomponent that reflects emitted fluorescent light from the substrate todirect it to a detector, rather than passing such light. The systems ofthe invention include a multi-band reflective dichroic filter thatselectively reflects multiple emitted fluorescent wavelength ranges,e.g., emitted by multiple different fluorescent materials havingdistinct emission spectra. In addition to their multi-band reflectivity,these filter components are typically capable of passing excitationlight (light at the desired excitation wavelength). As such, themulti-band dichroic are tailored to transmit excitation radiation atmultiple different wavelengths, while generally reflecting the longerwavelength emitted fluorescence. The dichroics are further tailored toinclude relatively narrow reflective ranges, so as to permittransmission of excitation bands that fall between or among two or moreemission bands. Such reflective fluorescence systems benefit fromsuperior performance dichroics, as compared to the transmissivedichroics, and also have cost and simplicity benefits.

Because the narrow-band selectivity is applied in reflection versustransmission, more of the reflected excitation radiation is filtered bybeing transmitted through the multi-band dichroic, and not reflected. Tothe extent that any excitation radiation is reflected by the multibanddichroic, it can be selectively filtered out following separation of theindividual excitation spectra (also referred to as ‘color separation’),using an individual narrow-band notch filter that is applied to oneseparated color (e.g., one selected emission spectrum), as all colors oremission spectra. As a result, any transmission losses are only appliedto an individual spectrum, and not over the entire emission spectra.Further, fabrication of a single multi-narrow band reflective filter ismore readily achievable using available technology than a narrowmulti-band transmissive filter.

FIGS. 8 and 9 provide schematic illustrations of conventionalfluorescence transmissive optical trains and the fluorescence reflectiveoptical trains of the invention. For ease of discussion, components thatare common among the two configurations are given the same referencenumbers. As shown in FIG. 8, a fluorescence transmissive optical train800 includes at least a first excitation light source, e.g., laser 806.For multi-band excitation, one or more additional light sources, e.g.,lasers 802 and 804 are optionally included. Where such additional lightsources are included, they are typically coupled with and directed atdichroic filters, e.g., dichroics 808, 810 and 812, respectively, sothat all of the excitation radiation from the various sources isco-directed, as indicated by the solid arrows. The excitation light isthen directed at a multiband dichroic filter 814 that reflectssubstantially all of the excitation radiation at the substrate 816 thatis being subjected to analysis. Fluorescent signals emitted from thesubstrate or sample surface are then passed through the multibanddichroic 814, which is transmissive to light at the wavelengths of theemitted fluorescence, along with some portion of reflected excitationradiation. In the case of multiple different fluorescent emissionspectra, the emitted fluorescence is then subjected to a colorseparation step, where the different individual emission spectra areseparated from each other and separately detected. Color separation maybe accomplished using a series of cascaded dichroic filters, such asfilters 818, 820, and 822 whereby a selected emission spectra isreflected from each of the dichroics onto an adjacent detector 832, 834and 836, respectively, with the last emission spectrum transmittingthrough all of the dichroics to be incident onto detector 838).Alternatively, a prism based color separation process may be employedwhere different emission spectra are directed through an appropriateoptical grating or prism to spatially separate the individual spectraand direct them to different detectors or different regions on an arraydetector. Additional filter elements, e.g., notch filters 824-830 may beincluded within the optical train to further tailor the emissionradiation detected at each of the detectors, e.g., to filter out anyinadvertent reflected excitation or emission light. As will beappreciated additional lasers, e.g., fourth fifth, etc. lasers, may beincluded in the system with the concomitant inclusion of additionaloptical elements, e.g., filters, dichroics, etc.

In contrast, FIG. 9 provides a schematic illustration of a fluorescencereflective optical train, in accordance with certain aspects of theinvention. As shown, although in a different orientation, the systemincludes similar excitation light sources (e.g., lasers 802-806) anddichroics (808-812) to codirect the excitation radiation. However, incontrast to FIG. 8, the excitation light is directed at and transmitted,rather than reflected by multi-band dichroic 902, which is tailored tobe reflective of multiple, narrow bands of emitted fluorescence. Theexcitation radiation is then transmitted, rather than reflected, bydichroic 902. Emitted fluorescence is then reflected, rather thantransmitted by dichroic 902, and then subjected to optional separationand detection, e.g., in a similar manner to that shown in FIG. 8. Aswill be appreciated, although the dichroics are shown oriented at 45°angles in the system to reflect light, e.g., fluorescence as in FIG. 9,at 90° angles relative to its angle of incidence, in some cases it maybe desirable to reflect the light at greater than a 90° angle, e.g.,rotating the dichroic so that the angle of incidence of both thetransmitted excitation light and emitted fluorescence is shallower than45°, as such higher reflectance angles provide for simplification indichroic fabrication.

The optical train included in the systems of the invention also mayinclude an autofocus function for automatically adjusting the objectiveor other lenses in the optical system to focus the sample material beinganalyzed within the focal plane of the optical train. A variety ofdifferent autofocus systems may generally be incorporated into thesystems of the invention.

As noted elsewhere herein, the optical trains of the invention, whetherbased upon fluorescence transmission or reflectance, typically directsthe emitted, and preferably separated, fluorescent signals to adetector. In particularly preferred aspects, the detector comprises anarray of point detectors, such as a diode array detector or a chargecoupled device (CCD, ICCD or EMCCD). In the case of such arraydetectors, it may be desirable for the optical train to provide thedirected fluorescence onto the detector in a particular desiredconfiguration. For example, in some cases, it is desirable to image afluorescent signal onto a plurality of pixels that exceeds a minimumthreshold level. For example, providing sufficient signal data from atleast 2 pixels, preferably at least 4 pixels, and more preferably atleast 10, 20 or even 100 pixels may be desirable to provide for enhancedstatistical evaluation of data. In accordance with this aspect of theinvention, the data from these multiple pixels will typically becombined before or during the processing of the signal data therefrom.In some cases, the signal data from the selected pixels would beaveraged and/or subject to correction, e.g., for background signal ornoise, in order to provide optimal statistical confidence in a givendata. In still other cases, the data may be combined prior tosubstantive processing, in order to reduce the data load that issubjected to the various processing steps, e.g., in a gain register orother processing system. In particular, and as described in greaterdetail below, like data, e.g., corresponding to a single signal or tobackground or quiet pixels, may be co-processed in order to minimize theamount of individual data units that are subject to such processing, andthus reduce the processing requirements of the overall system.

In the case of signals having multiple, separated spectral components,it may be desirable to image each different fluorescent signalcomponent, e.g., each differently colored spot of emitted fluorescence,onto a plurality of pixels of an array detector, so that variations inintensity across an individual signal spot may be accommodated in dataanalysis, e.g., averaged, discarded, etc. For example, in many cases,each signal component will be imaged on at least two pixels in adetector array, preferably at least 4 or more pixels in the detectorarray, and in some cases upwards of 10, 20 or 50 or more pixels.

In the case of signal sources, e.g., sample substrates that include anarray of discrete signal sources, the total number of pixels involved indetection of a given spectral signal from the overall array willtypically vary approximately by the multiple of the sources beinganalyzed. For example, if each separated color signal from each discretesignal source on an array is imaged onto 4 pixels in the detector array,and 10 signal sources were being analyzed using the same array, then theaggregate signal for that color for the entire array of signal sourceswould be imaged onto approximately 40 pixels of the detector array. Ashas been reiterated herein, in particularly preferred aspects, theimaged signal will typically include at least two separated spectralcomponents, and preferably 3, 4 or more spectral components that aredirected to and imaged upon different detectors or regions on a detectorarray, utilizing a range of numbers of pixels.

V. Detector

The systems of the invention may generally include any of a variety ofdifferent detector types useful for detecting optical signals that aredirected to the detector. Examples of different types of detectorsinclude photodiodes, avalanche photodiodes, photomultiplier tubes,imaging detectors, such as charge coupled devices, CMOS (complementarymetal oxide semiconductor) sensors or imagers, CCD/CMOS hybrid imagers,and the like. In preferred aspects, imaging detectors are employed inthe systems of the invention, so as to provide simultaneous detectionover larger areas of the substrates, and consequently, larger numbers ofdiscrete signal sources. Charge coupled device based detectors (CCDs)and CMOS image sensors are particularly preferred for their ability tosimultaneously detect and/or monitor signals from large numbers ofdiscrete signal sources on the substrate. Because data derived fromthese types of image or imaging detectors is assigned to discretepixels, signals from discrete sources that are incident upon differentlocations of the detector may be separately detected and quantified.Further, in applications where relatively high speed, and relatively lowsignal levels are prevalent, e.g., where the signal sources comprisesingle molecule type reactions, highly sensitive detectors are generallypreferred, such as electron multiplying CCDs (EMCCD) or intensified CCDs(ICCD). Typically, EMCCDs are preferred for their sensitivity to lowsignal levels.

FIG. 10 provides a schematic illustration of the operation of anexemplary EMCCD in processing image data. As shown, an overall system1000 includes a typical EMCCD chip 1002, which has an image area 1004and a storage area 1006. The CCD includes an EM gain register 1008 thatis operably connected to an appropriate analog:digital converter 1010,which is, in turn, connected to a processor or computer, e.g., computer1012. As shown, each area comprises a 512×512 pixel array. As shown instep A, an image is acquired (Step A) (Frame 1) in the image area 1004and transferred to the storage area 1006 (step B) so that the image areais available for acquiring subsequent images, e.g., Frame 2 (Step C). Inthe case of some EMCCDs, the frame transfer requires an appliedpotential of approximately 2V. The frame in the storage area (Frame 1,as shown) is then transferred into the EM Gain register 1008 pipeline(step D) (again, requiring approximately 2V), where the chargeassociated with the image is passed through approximately 536 stages toachieve a potential gain range, that is software controllable, from 1 toof 2000×. The EM gain register processing typically requiresapproximately 50V. The amplified image data is then passed through ananalog to digital converter 1010 (step E) to be stored or furtherprocessed by a computer 1012 (step F).

As with the illumination of signal sources, in preferred aspects, thedetection systems in the systems of the invention are typically capableof detecting and/or monitoring signals from at least 2 different signalsources, simultaneously, preferably, at least 10 discrete signalsources, and in many cases, more than 100 or even more than 1000discrete signal sources, simultaneously. Further, the detectors arelikewise capable of monitoring or detecting multiple, spatiallyseparated signals or signal components from each such source. Inparticular, as noted above, signals from each discrete source arepreferably spatially separated, at least partially, into at least two,and preferably, three, four or even more separate signal components,that are directed onto the detector array and are capable of resolutionand ultimately being separately detected. In some eases, two differentsignals that may be emitted from a given signal source may not becompletely spatially separable onto different regions of a detectorarray. However, because such signals differ in their emission wavelengthspectra, subjecting such different signals to the wavelength separationcomponents of the optical train, e.g., a prism such as prism 610 in FIG.6, can yield imaged signals on a detector array that have imaged shapesthat are characteristic of the particular emission spectrum, while notbeing completely spatially separable from another signal componentshaving slightly different emission spectra. In such cases, identifyingthe signal component that gives rise to a detectable event can sometimesinclude identification of a characteristic shape of an aggregate groupof pixels upon which such signal is incident. As will be appreciated, inthose cases that utilize detector arrays as image detectors, e.g., CCDs,CMOS sensors, and the like, detection of image shape will typicallyrefer to detection of signals at the various detector elements, orpixels, that are reflective of an imaged signal of a given shape. Thus,identifying a signals imaged shape will generally refer to detection ofsignal at pixels underlying that image shape, rather than holisticallyidentifying the shape. Further, the identification of the signalcomponent based upon the imaged shape may not specifically include astep where the shape is identified, but rather that signal is detectedthat is characteristic of that shape. Thus, with respect to thesemethods, identification of image shape may not include any step wherebythe shape is actually identified, e.g., “shape is circular”, but mayonly be identified by the identification of the pixels upon which thesignal is incident.

VI. Data Management

The systems of the invention also typically include a data processingsystem coupled to the detector for processing and/or recording signalsthat are incident upon and detected by the detector, and for processingthat data to useful information for the user. For example, in the caseof single molecule analyses, e.g., where the signal source comprisesfluorogenic reactants, the data processing system may assign a value tothe incidence of signal on a given location of the detector at aparticular time, as being indicative of the occurrence of a givenreaction. The data derived from each signal would typically include oneor more of (a) the intensity of the signal, (b) the pixel or pixels uponwhich the signal was incident, (c) relative time that the signal wasdetected, and the like. Such data may then be processed to indicaterelative rates or activities of reactants, order of reactions, aparticular signal source from which the signal was derived, and throughknowledge of that source's reactants, the nature of an analyte exposedto such reactants.

For ease of discussion, where the signal source includes templatedirected DNA synthesis using fluorescent nucleotide analogs and DNApolymerase enzyme within an optical confinement, a signal may beindicative of the incorporation of a nucleotide at a given relativeposition in the synthesis. Further, using the spectral separationaspects of the optical train, and four different nucleotide analogs allbearing dyes or labels having resolvably different spectralcharacteristics, e.g., that are separated by the optical train anddirected to different locations on the detector (or that possessdifferent imaged shapes) as a result of their differing spectralcharacteristics, a signal at a given location on the detector (or havinga given shape) can be indicative of incorporation of a specific type ofanalog, and the relative timing of such signal would be indicative thatsuch base occurs in the template sequence before or after another basewhich gave rise to an earlier or later signal, respectively. Finally,the location on the array where such signals are incident is indicativeof the signal source from which the signals derive (e.g., indicatingthat subsequent signals at the same approximate location (subject to,e.g., spatial separation based upon spectral differences of componentsof signals from a given source) are likely a result of the continuationof the same reaction). This detection is repeated multiple times toidentify the sequence of incorporation of multiple nucleotides. Byvirtue of the complementarity of incorporation in template directed DNAsynthesis, one may then ascertain the underlying sequence of nucleotidesin the template sequence.

In at least one aspect, as with the aspects of the invention that adjustthe array of sources depending upon expected optical aberrations, onemay also adjust the methods by which data is acquired and/or assigned toindividual sources, based upon those expected optical aberrations. Inparticular, as noted previously, an amount of distortion of an imagedarray can increase as a function of distance from the axial center ofthe object field. As a result, correlating or assigning individualpixels or groups of pixels to a specific signal source in an imagedarray becomes more difficult away from the center of the image.Additionally, optical aberrations may further deform the shape of theimaged signal depending upon the position on the detector array of theimaged signal. For example, certain optical aberrations, i.e., coma, mayyield an imaged signal from a circular source that is ‘tear-drop’shaped, falling away from the axial center of the imaged field.Alternatively, combinations of astigmatism and field curvature couldresult in an elliptical signal image shape from a circular signalsource, which is more pronounced with increasing distance from the axialcenter of the object field.

Accordingly, in at least one aspect, one can accommodate increasinglevels of distortion by expanding the number of pixels that arecorrelated to any given source, in conjunction with a known or expectedoptical aberration of the system. In a simple form, this involvesincreasing the number of pixels correlated to a given source beingimaged as that image (or its respective image source) is farther awayfrom the center of the image or object field. A schematic illustrationof this is shown in FIG. 11. As shown, an array of pixels in an arraydetector, e.g., a CCD 1100, is provided to image the array of signalsources. As shown in panel A, in the absence of optical aberrations,uniform signal sources yield uniform images upon the CCD, e.g., asindicated by signals 1102-1112, regardless of where in the image fieldthey emanated from. However, in the case of systems sensitive to suchoptical aberrations, as the distance increases between the center of theimaged field and a given imaged source, e.g., moving from imaged spots1120 and 1122 to spot 1124, the distortion results in increasing imagesize, and/or lower resolution. In order to account for this distortion,the pixels correlated to a given image or signal are increased tomaximize the data acquired for each imaged signal, e.g., by acquiring asmuch of the given signal as possible or practicable, e.g., including allof the different pixel regions at the center and periphery of the objectfield, imaged onto the CCD. The adjustment of correlated or recordedpixels for any given signal image is a particularly useful process whencombined with an array of sources that is further arranged to accountfor such optical aberrations, e.g., see FIG. 3, above. Alternatively, oradditionally, and also as shown in FIG. 11, one may adjust the assignedpixels for a particular imaged signal to account for distortions in theshape of the imaged signal, e.g., for an elliptical or tear-drop shapedimage. In particular, one may employ a collection of pixels for anindividual imaged signal that is larger in one axis than the other,e.g., longer in the y axis as shown in FIG. 11.

In addition to the improved ability to separately monitor signals fromdiscrete sources, the use of such CCD or other array detectors providesadditional benefits for analysis of signals from the individual signalsources as well as the aggregate signals from the overall array ofsignal sources. For example, where a signal from a given discrete sourceis incident upon multiple pixels, the compartmentalization of data on apixel basis allows selection of optimal pixels in a given imaged signal,for data analysis, e.g., eliminating edge signals that may have higherlevels of noise or distortion. Additionally or alternatively, pixelsused to obtain signal data for each discrete signal source may beindividually tailored for a variety of different purposes, as discussedelsewhere herein. The management of such pixel data is further describedin greater detail below.

In addition to accommodating and/or correcting for optical aberrations,the present invention also provides processes that provide moreefficient processing of relevant signals. In at least one generalaspect, such processes involve the further processing of only relevantsignals, while either discarding or combining less relevant signals. Ineither case, by reducing the amount of signal data that is subjected tothe full range of further processing, one can speed up that processing,reduce processing requirements, e.g., computing power, reduce realestate on an array detector required for image data management, extendthe lifespan of detector components, and achieve a variety of otherbenefits. These processes generally may be carried out either in thecontext of the CCD chip, or they may be performed in a subsequent,off-chip processes, e.g., using a computer. As will be appreciated, inmany cases, preferred implementations are carried out within the imagedata processing steps on the detector array itself.

In the context of the present invention, it will generally be understoodthat the term “processing” refers to automated processing of data by amechanical or solid state processor or system that is programmed tocarry out such processes, e.g., in machine readable software orfirmware. Thus, the processing steps may be carried out by a singlesolid state device, e.g., an appropriately configured detector chip suchas an EMCCD, or by a connected or integrated computer or otherprocessor.

As alluded to above, in certain aspects, the invention provides for aninitial data processing or selection step to avoid the management,storage and/or processing of excessive irrelevant data that is or wouldbe produced by the detection system, as well as the combined processingof certain data from different areas on the detector. In particular, insome cases, one may gain significant advantages, e.g., in terms of speedof data processing and management and usefulness of background signaldata, through the selective skipping, removing, or combining of pixeldata prior or subsequent to extraction of data, e.g., from a CCD chip.Stated in another way, by ignoring or separately processing datacollected from certain pixel areas that do not contain highly relevantdata, e.g., they fall outside of a relevant imaged signal, one can speedup the data management process by removing large amounts of irrelevantdata from the process or combining into one processable unit, all of thebackground or less relevant signal data. Additionally, or alternatively,such combined less relevant pixel data may be useful to derive moremeaningful background signal levels, or noise, of the system. In eithercase, the speed and accuracy of the system should benefit.

By way of example, where one is imaging a large number of discretesignal sources or separated signals derived from such sources, on asingle detector array, e.g., a CCD, ICCD or EMCCD, space between imagedsignals from such discrete sources gives rise to little or no usefuldata, as it is a “quiet” space. Notwithstanding the lack of usefulsignal data emanating from these regions of the detector array, the datafrom such locations has typically been recorded, e.g., as a zero, orsome other low level signal value, or other irrelevant value. While suchsignals can be disregarded as background, their recordation andprocessing to the point of discard still requires memory space forstorage and processing capacity for evaluation and ultimate discard.Accordingly, in certain aspects, the invention provides a maskingprocess for filtering out such quiet locations on the detector array,and thus blocking the data from being recorded.

For example, in a first aspect, rows of detector array elements, such aspixels in CCD based detectors, that fall between rows of imaged signalsfrom the discrete signal sources, and thus carry signals that are not asrelevant to the desired analysis, may be skipped during data extractionfrom the detector arrays. FIG. 12 provides a schematic illustration ofthis data extraction profile in a CCD array. As shown, individualsignals 1204 from signal sources (not shown) are imaged onto an arraydetector, e.g., CCD 1202. As shown, the imaged signals 1204 are imagedupon rows of pixels 1206 that are interspersed with rows of pixels 1208upon which no relevant signals are being imaged, also generally referredto as “quiet pixels”. As will be appreciated, within each row of pixels1206 upon which are imaged relevant signals, there may exist quietpixels between each individual imaged signal element, e.g., pixels 1210.For ease of illustration and discussion, the extraction of data frompixel rows and/or columns is generally illustrated with respect to pairsof adjacent rows and/or columns, rather than from individual pixel rows,but such illustration is not indicative of any process requirement orother parameter.

In a typical image extraction process, all of rows 1206 and 1208 wouldbe subjected to the same processing steps, resulting in a substantialamount of resources being dedicated to the processing of the lessrelevant or quiet pixels. This is schematically illustrated by thearrows emanating from each pixel row (or pair of pixel rows, as shown),e.g., relevant signal rows 1206 and quiet pixel rows 1208.

In accordance with certain aspects of the invention, and as shown in theimage in panel B, however, data is extracted from the pixels, e.g., therows and/or columns that carry imaged signals, e.g., rows 1206, from anarray of signal sources, while the intervening rows and/or columns,e.g., rows 1208 (and optionally quiet pixel columns that include, e.g.,pixel regions 1210) are ignored from a data extraction standpoint. Thisis shown in FIG. 12, panel B.

In particular, as shown, an application of the process would involveskipping extraction of data from rows 1208, while extracting data fromrows 1206. While data from the analyzed rows is subjected to furtherprocessing, e.g., passed through EM gain register and/or theanalog-digital converter (ADC), to the computer or processor forsubsequent storage and manipulation, the skipped rows are not. Thiseffectively reduces the amount of data that is run through the ADC bymore than half, in the example shown. Alternatively or additionally, thedata derived from rows 1208 may be separately combined and/or averagedprior to or subsequent to extraction (shown by the dashed arrow in panelB) to provide a more significant determination of background noiselevels of the system, which may then be used to further correct thesignal data extracted from, e.g., rows 1206. Even with such processingof the quiet pixel data, by binning this data together for processing ina single processable data unit, the efficiencies described above arelargely retained.

In other aspects, data from related array elements may be combined or“binned” before being subsequently processed, in order to minimize thenumber of separate data elements that are subject to processing. Forexample, with reference to the extracted row data described above, eachset of rows and/or columns that corresponds to a particular signalsource image, or the space between imaged signal sources, may beseparately binned for subsequent processing, reducing the number of dataelements that are subjected to processing. Similarly, pixelscorresponding to images from individual signal source array elements maybe binned together and processed. In each of the foregoing cases,whether alone or in combination, the overall number of data elements issubstantially reduced over the extraction and processing of eachindividual pixel element.

In addition to providing benefits of data management selectively binningpixels of imaged signal components may provide advantages of dataanalysis. For example, when imaging spatially separated signalcomponents, one can selectively bin those elements that are derived fromsignal rows that are of similar fidelity, allowing subsequentidentification of lower fidelity signals, in aggregate. As notedpreviously, in certain embodiments, the constituent elements of eachsignal, e.g., the different signal wavelengths emanating from eachsignal source, are subjected to spatial separation and are imaged ontodifferent pixels, or collections of pixels, on the detector array. Aswill be appreciated, because constituent signal wavelengths tend to fallover a range rather than within a precise single wavelength orwavelength range in some cases, and because addition of more signalwavelength components within the signal sources as may occur withvarious applications and/or multiplexing, spatial separation may yieldless than complete separation between different signal constituentsalong each row, e.g., resulting in spectral overlap of the separatedsignals.

In accordance with certain aspects of the invention, data that is ofhigher fidelity is processed separately than lower fidelity data, evenwithin an imaged signal. In its simplest sense, only pixels thatcorrespond to the highest fidelity data, e.g., having the highestintensity relative to a noise level of the system, are processed asrelevant signals. Other signal components are then subjected todifferent processing or are discarded. In general, as will beappreciated, such signal components are those that are within the mainportion of the imaged signal, e.g., toward the center of the imagedsignal, rather than at its periphery. An example of this is illustratedwith reference to FIG. 13A which shows a representation of an imagedsignal 1300 upon a set of pixels 1302 in an array detector. Inaccordance with the signal selection processes described herein, onlythose signals derived from pixels at or near the central portion of theimaged signal, e.g., pixels in region 1304 (shown without hatching) aresubjected to processing as relevant signal data. Signals from pixels atthe periphery of the signal, e.g., pixels in region 1306 (shown crosshatching), would be expected to be of lower fidelity, e.g., having lowersignal to noise ratios. Accordingly, pixels in region 1304 are subjectedto processing as relevant signal while pixels in region 1306 are treatedseparately which may include discarding or inclusion in determination ofan overall system signal to noise ratio. As will be appreciated, theselection of higher confidence signal data or their respective pixelsmay be carried out by a number of parameters including withoutlimitation, selection of higher intensity signals within an overallimaged signal, and/or selection of signals that are expected to be ofhigher confidence based upon their position in an overall imaged signal,e.g., they fall within a central portion of the overall imaged portion,where the central portion refers to a signals from a subset of pixelsimpinged upon by the overall imaged signal, while pixels that are withinthe overall imaged signal, but fall at the periphery or around theentire edge of the imaged signal, are discarded. For generality, itcould be viewed that the signal portion that extends only a portion ofthe radius of the overall imaged signal, would be viewed as of highconfidence, where that portion may vary from, e.g., 25%, to 50% to 75%or even 90% where signal images are highly coherent.

A more complex implementation of this selection process, where a signalfrom a given source is spatially separated with incomplete separation,e.g., with substantial signal overlap, is shown in FIG. 13B. As shown, asignal is imaged upon a set of pixels 1320 in an overall detector array.As shown, the signal is subjected to spectral separation whereby signalcomponents having different spectral characteristics are directed todifferent (albeit overlapping) groups of pixels on the array. This isillustrated by signal images 1322, 1324, 1326, and 1328 which showconsiderable overlap. In accordance with this aspect of the invention,less relevant pixels, such as those that are at the periphery of eachsignal component or are occupied by overlapping signals, such as pixels1330 (shown cross hatched), are discarded prior to, or combined forprocessing. Meanwhile, high fidelity signals upon, e.g., pixels 1332(shown without hatching) are subjected to further processing as relevantsignals.

In accordance with the processing aspects of the invention, relevantdata, e.g., from pixels 1332, from each signal, e.g., signals 1322-1328,can be binned together for each signal component and processed as shownby the dashed arrows, e.g., passed through the EM gain register, the A/Dconversion, and subsequent processing by the computer. All other, lowerfidelity data surrounding the signals, as well as that which is includedin the signal overlap regions (e.g., pixels 1330, may be discarded orbinned together for simultaneous processing, e.g., A/D conversion,inclusion in background signal calculation, etc.

By binning the lower fidelity data, e.g., that includes excessive levelsof mixed signal constituents, one can effectively discard or process allof these signals simultaneously, or at least separately from therelevant pixel data. In accordance with certain aspects of theinvention, the data is binned in a manner that combines each set ofpixels that includes the same level of spectral overlap (or absencethereof), as shown by arrows 1334 and 1336. As with the quiet detectorspaces referred to previously, data from the pixels that fall betweenthe pixels having the highest fidelity signals may be processedseparately from the high fidelity signal data. For example, it may bediscarded prior to subsequent processing, or it may be binned andprocessed in merely a separate process operation from the high fidelitydata. Alternatively, it may be combined with all other low fidelitydata, to generate a background level of spectral overlap signal, or thelike.

In accordance with the foregoing and other aspects of the invention, itwill be appreciated that rows or columns of pixels may include rows orcolumns that range from a single pixel width to 2 or more, 5 or more, 10or more, or 100 or more pixels in width, or any pixel width that fallswithin these ranges. The specific number of pixels that fall within agiven row or column, whether it be a signal row or column or a “quiet”row or column, will depend upon the desired application, and they may bevaried from system to system, or even within a given system, e.g.,column and row widths in the monitoring of any given substrate may varyacross the detector, e.g., one signal row may be two pixels wide whileanother row is 10 pixels wide. Likewise, in the same application, whilea given quiet row may be 2 or 10 pixels wide, another quiet row in thesame detection event may be 10 or 20 pixels wide.

Further, any of these signal data manipulation techniques may be applieddynamically, to optimize different parameters, e.g., signal to noiseratio, for each analytical operation that is being performed. Inparticular, one could adjust the relative spacing of the excluded rowsand/or columns, the number of pixels being assigned to each signalevent, or any combination of these to achieve a desired signal to noiseratio, e.g., by comparing a standard signal to a background noise.Further, this could be performed using appropriate software programmingto be able to optimize for any of a number of different regions ornumbers of regions or signal sources imaged onto an array.

In some cases, it may be desirable to provide a physical mask over anarray detector to filter any signal derived from areas between thesignal sources spaces on the detector array to filter out any noisederived from signal in adjoining signal sources/pixel areas. Thephysical mask may comprise a separate optical element, e.g., an opaquesubstrate having optical apertures disposed at regions that correspondwith the imaged signals, e.g., similar to photolithographic masks usedin semiconductor fabrication. Alternatively, the mask may be provided asa layer over the detector array, e.g., using light absorbing polymers orpolymers containing light absorbing materials, photoresists, or thelike.

As will be appreciated, noise that derives from the system itself, andthat will still be present in the event that a mask is used withoutother adjustments, may be accounted for and dealt with in any of themethods described above. In a further aspect, one could employ detectorarrays that are specifically configured, e.g., through the placement ofdetector elements, e.g., pixels in a CCD, to correspond to the regionsupon the array where signals will be incident, and thus excludebackground signal events, e.g., that would be incident on the arraybetween relevant imaged signal events.

In at least one aspect of the invention, a modified EMCCD is used as thedetector array. In particular, and as schematically illustrated in FIG.10, conventional EMCCDs use a frame transfer process in moving data tothe storage area of the CCD chip, and then use a separate EM Gainregister to provide signal gain of up to 100×, 500×, 1000×, 2000× ormore before the data is digitized and transferred to the processor,e.g., a connected computer. While this process is effective in thedetection of low light level signals, the separate EM Gain register canbe quite large, relative to the overall chip footprint, occupying agreat deal of CCD chip real estate. In accordance with certain aspects,the EMCCD is configured so that the clocking voltages used for the rowshift process are arranged to realize the gain during the transfer ofdata from the image area to the storage area, rather than post storagevia an EM Gain register. In particular, as noted previously, typicalframe transfer process to the storage area on the CCD chip, andsubsequent transfer to the EM gain register are each carried out with anapplied potential of approximately 2V. Processing the charge associatedwith the signal through the gain protocols in the EM gain register isthen done with an applied potential of approximately 50V. By applyingthe 50V and implementing the gain protocol during the frame transferprocess, one can obviate the need for the EM gain register.

In addition to the foregoing, and as further examples of the benefits ofthe invention, current EMCCD cameras operate by adding a long string of“pixels” (several hundred) and applying a very high voltage (50V ormore) to move the data from one pixel to the next. 50 V is sufficient tocause a small probability of creating spurious charges—for example ifone electron is being moved from one pixel to the next, there is a 1%chance that an extra electron will be created, thus doubling theapparent signal strength. Simple statistics can be used to show that again of 1000× can be achieved with a 1% probability per pixel andapproximately 400 pixels. The drawback of this approach is that there istypically only one gain amplifying channel for the entire EMCCDchip—this means that data from every single pixel must be funneledthrough the same gain amplifier. In a particular exemplary EMCCD camera,the data is passed through this single gain amplifier and then digitizedat a rate of 10 Megahertz, meaning a maximum frame rate of the camera is33 Hz (512×512 pixels divided by 10 Megahertz).

In the context of the invention, however, applying a higher voltage tothe frame transfer process, e.g. similar voltage level to that used inthe gain amplifier of conventional EMCCDs, one could attain similar orgreater amplification. Further, and with reference to an exemplary EMCCDchip having 512 rows of pixels (512×512), the frame transfer processwould include 512 transfers from one pixel to the next. A voltage lessthan 50V, with a probability a bit less than 1%, would provide the 1000gain that is available through a integrated gain register. As a resultof negating the need for a gain register and its associated bottle neckand chip area requirements, the EMCCD according to the instant inventionwould be much faster, and the real estate required could be about half,which would be expected to cut the chip cost in half.

Thus, in certain aspects, the invention includes methods for processingimage data from a CCD, and particularly an ICCD or EMCCD, that includeapplying gain voltage during a frame transfer process, and CCD baseddetectors that are configured to carry out this process. By utilizingthe frame transfer process as the gain amplification process, onesignificantly increases the processing speed for signal data,significantly reduces the chip real estate, and consequently the chipcost associated with a typical EMCCD camera or other such detector.

Another aspect of the signal image data processing aspects of theinvention provides benefits in terms of preservation of systemperformance, in addition to providing advantages in efficiency of dataprocessing. In particular, as noted above, CCD detectors, andparticularly, high sensitivity CCDs, such as EMCCDs are preferred foruse as detector arrays in the systems of the invention, because they canoffer a combination of high gain, parallel readout and fast framerate.These attributes make such detectors particularly well suited for use inapplications that are temporally monitoring operations that yield verylow level optical signals, such as single molecule analyses. However, asa result of possessing these attributes, the EMCCDs may be subject todegradation of performance. In particular, in the case of EMCCDs, the EMgain register may be subject to rapid degradation when large amplitudesignals are passed trough it.

In preventing such degradation, it is generally desirable to limit theamplitude of signals being processed by the EM gain register. However,even limiting such amplitudes to within manufacturer recommendationsstill can yield substantial degradation. Without being bound to aparticular theory of operation, it is believed that a contributingfactor to gain degradation is the combination of signals on the CCDchip, when a subset of pixels of the array in a region of interest on anarray is read out from the chip. This operating mode can be implementeddifferently on different EMCCD configurations, but for at least someconfigurations, the pixel rows that are outside of the region ofinterest are combined together and then passed through the EM gainregister. This can lead to substantial variation in the amplitude of thesignals being passed through the register, leading to degradation.Accordingly, the present invention also provides methods for reducing oreliminating the large amplitude variation of signals being processed bythe gain register.

In certain aspects, this is achieved applying the processes describedelsewhere herein. In particular, the excess charge that derives fromregions of the CCD that are not used to image relevant signals, e.g.,those regions of the CCD that fall between or outside imaged signalsfrom signal sources, are cleared before passing the overall signalsthrough the gain register. In particular, a number of EMCCDconfigurations are available e.g., EMCCDs from E2V Technologies, Inc.,that include electrical taps that may be used to bypass the gainregister and send the signal output from these other regions to aseparate destination, e.g., as shown by the dashed arrow in FIG. 12,panel B, but leading to a separate output from the EM gain register.These taps may be configured on a per frame basis, and may, along withthe control pins, be integrated into the CCD design.

In an alternative or additional method, and as alluded to above, one mayalso only subject certain highly relevant array regions to subsequentprocessing, e.g., disregarding regions where little or no relevantsignal is imaged, e.g., spaces between or surrounding relevant signalregions on the array. By avoiding passing data from these regionsthrough the EM Gain register, at least one source of large amplitudevariations, e.g., the variation from relevant signal bearing pixels andpixels that are just communicating noise, in the signal data can beavoided. This is generally accomplished by providing for a readout ofthe chip on a segmented basis, e.g., pixel by pixel, or sub-region bysub-region. This is a particularly useful solution where the imagingframe rate is not required to be fast, e.g., greater than 33 Hz,allowing for the slower processing methods.

For higher framerate applications, the charge combination effect can bemitigated by reducing the number of rows that can be combined together.For example, and with reference to FIG. 14, in a 512×512 pixel EMCCD1402, where within the image area 1404, an 80×80 sub-region 1406 is readout, over up to 432 rows of irrelevant or quiet pixels 1408 can becombined together. If these rows are combined in groups of ten, insteadof as a single group, the damage effect will be reduced by a factor of43, while maintaining a relatively high framerate, e.g., 100 Hz orgreater.

In yet another aspect, a CCD may be programmed to adjust the EM gain,dynamically as it recognizes large signal amplitude variations. In suchapplications, the voltages of the EM gain register would be reduced whenprocessing charge from outside of the region of interest on the array.Alternatively, software can be implemented to monitor the maximumsignal, and reduce the EM gain automatically when that signal exceeds aproscribed level. This is schematically illustrated in the process flowchart provided in FIG. 15. This can also be extended to signal packetsthat come through the EM gain register. Finally, masking techniques,e.g., knife edge masks, can be employed to mask off rows on the CCD thatare outside of the regions of interest that would otherwise be combined.

Relatedly, the detectors and/or systems of the invention may provide forthe automatic measurement and/or calibration of the gain, byautomatically determining the gain in the absence of actual signals. Inparticular, in many multiplying image detectors, such as EMCCDs,degradation of gain over time can create issues of signal and dataquality, unless the gain is regularly measured and calibrated, so as toprovide amplified signal data within a desired range. In the past, thismeasurement has been carried out manually, requiring significant timeand effort, and introducing potential avenues for human variation intoan overall process.

In accordance with this aspect of the invention, the gain is measuredduring a period where no signal data is incident upon the detector.Typically, this may be accomplished by automatically closing the shutterof the optical system so as to block signal data from impinging on thedetector. Likewise, this could be accomplished by turning off any lightsources that might provide such signals. In any event, the gain measuredin the absence of signal is then used to calibrate the gain register soas to fall within a desired gain range, and/or to provide signal datathat will fall within a desired amplified signal range. These processesmay generally be programmed into the controlling computer, e.g., thatinitiates closure of the shutter, records measured gain and recalibratesgain register.

Although described in some detail for purposes of illustration, it willbe readily appreciated that a number of variations known or appreciatedby those of skill in the art may be practiced within the scope ofpresent invention. To the extent not already expressly incorporatedherein, all published references and patent documents referred to inthis disclosure are incorporated herein by reference in their entiretyfor all purposes.

1.-83. (canceled)
 84. A method comprising: providing a substratecomprising a plurality of discrete sources of optical signals;illuminating the plurality of discrete sources with an excitation lightsource, whereby emitted light is emitted from the plurality of discretesources, the emitted light corresponding to signals from the discretesources; directing the emitted light Onto a detector comprising an arrayof pixels, wherein for each discrete source, a subset of pixels thatcorrespond to an image of that discrete source is designated as a set ofhigh fidelity pixels, and other pixels on the detector are designated aslow fidelity pixels; combining the data from the pixels within each setof high fidelity data for each discrete Source; and processing thecombined data to provide information about the signals from the discretesources.
 85. The method of claim 84 wherein data from the low fidelitypixels is combined.
 86. The method of claim 85 wherein the data from thelow fidelity pixels is discarded.
 87. The method of claim 84 wherein thedata from the low fidelity pixels is used in determining the backgroundnoise level.
 88. The method of claim 84 wherein the array of pixels isoriented in rows and columns, and wherein all of the pixels in some ofthe rows and all of the pixels in in some of the columns are designatedas low fidelity pixels.
 89. The method of claim 88 wherein the rows orcolumns range from 2 to 20 pixels wide.
 90. The method of claim 84wherein the images of set of high fidelity pixels corresponds to thecentral portion of the image of the discrete source.
 91. The method ofclaim 90 wherein the high fidelity signal corresponds to about 25% to90% of the radius of the image of the discrete source.
 92. The method ofclaim 84 wherein the pixels designated as high fidelity and as lowfidelity is adjusted dynamically.
 93. The method of claim 84 wherein thedesignation of high fidelity and low fidelity pixels is adjusted tooptimize for different regions on the array.
 94. A system comprising: asubstrate comprising a plurality of discrete sources of optical signals;an optical train configured to direct excitation radiation from anexcitation source to the plurality of discrete sources; receive emittedoptical signals from the array of signal sources; and image the emittedoptical signals onto a detector comprising an array of pixels, whereinfor each discrete source, a subset of pixels that correspond to an imageof that discrete source is designated as a set of high fidelity pixels,and other pixels on the detector are designated as low fidelity pixels;and a first processor configured for combining the data from the pixelswithin each set of high fidelity data for each discrete source; and asecond processor configured for processing the combined data to provideinformation about the signals from the discrete sources.
 95. The systemof claim 94 wherein the detector comprises the first processor.
 96. Thesystem of claim 94 wherein the detector comprises a CCD, an EMCCD, or anICCD.
 97. The system of claim 94 wherein the first processor and thesecond processor are on a computer that is separate from the detector.98. The system of claim 94 wherein the first processor or secondprocessor is configured to combine, data from the low fidelity pixels.99. The system of claim 94 wherein the first processor or secondprocessor is configured to discard data from the low fidelity pixels.100. The system of claim 94 wherein the first processor or secondprocessor is configured to use the data from the low fidelity pixels todetermine the background noise level.
 101. The system of claim 94wherein the array of pixels is oriented in rows and columns, and whereinall of the pixels in some of the rows and all of the pixels in in someof the columns are designated as low fidelity pixels.
 102. The system ofclaim 101 wherein the rows or columns range from 2 to 20 pixels wide.103. The system of claim 94 wherein the images of set of high fidelitypixels corresponds to the central portion of the image of the discretesource.
 104. The system of claim 94 wherein the high fidelity signalcorresponds to about 25% to 90% of the radius of the image of thediscrete source.